Multimicrolamellar Collagen Membranes

Abstract

The present invention relates to multimicrolamellar membranes of collagen of several types and structures (e.g. collagen type I, type II, type III, etc.), of only one type or two types in association, but more particularly of type I and type II collagen and/or of their mixture. The multimolecular arrangement is obtained by preparing the membrane in several steps involving the sequential addition of collagen gel layers. Membranes look thin, with a variable rough surface depending on the type of collagen used. Such membranes show a parallel horizontal lamellar microstructure and they can be soaked with different fluids, act as three-dimensional scaffold for cultured cells and are apt to be infiltrated and colonized by cells in vivo, therefore working as support suitable for direct, guided tissue regeneration. Moreover, the present invention relates to the preparation of said membranes from collagen gels and to the optimization of the preparation of collagen gel from tissues.

1 . Collagen membrane composed of at least 2 layers, wherein each layer is composed of a single type of collagen or a mixture of different types of collagens, characterized by a multimicrolamellar structure under scanning electron microscope. 
   
   
       2 . Membrane according to  claim 1  wherein at least one layer is type I collagen. 
   
   
       3 . Membrane according to  claim 1  wherein at least one layer is type II collagen. 
   
   
       4 . Membrane according to  claim 1  wherein at least one layer is a mixture of type I and type II collagen. 
   
   
       5 . Membrane according to  claim 4  wherein at least two layers are a mixture of type I and type II collagen. 
   
   
       6 . Membrane according to  claim 5  wherein said mixture contains type II collagen in a proportion comprised between 10-70% of the total collagen. 
   
   
       7 . Membrane according to  claim 6  wherein type II collagen is in a proportion comprised between 20-60%. 
   
   
       8 . Membrane according to  claim 7  wherein type II collagen is in a proportion comprised between 30-50%. 
   
   
       9 . Membrane according to  claim 5  having an imbibition capacity of at least 100% of its dry volume. 
   
   
       10 . A process for the preparation of membranes from collagen gels according to  claim 1 , comprising the following steps:
 a) electrostatic loading of a tray by its interposition in a vertical electrostatic field of at least 100 kv/m,   b) pouring a collagen gel in an electrostatically loaded tray and further interposition of the gel-containing tray in an electrostatic field of at least 100 kv/m with vertical orientation;   c) exsiccation of the collagen gel layer up to a residual water content of at least 30%,   d) pouring of at least one additional collagen gel layer;   
   
   
       11 . The process according to  claim 10  wherein, prior to electrostatic loading, trays are leant against a dispersion surface or otherwise electrostatically discharged. 
   
   
       12 . The process according to  claim 10  wherein the tray is electrostatically charged prior to pouring each subsequent layer of collagen gel. 
   
   
       13 . The process according to  claim 10  wherein the electrostatic field has a power comprised between 100-220 kv/m. 
   
   
       14 . The process according to  claim 10 , wherein the tray is kept in the electrostatic field for at least 2′. 
   
   
       15 . The process according to  claim 10  wherein each collagen gel is flattened by use of a spatula. 
   
   
       16 . The process according to  claim 10  wherein in addition the following conditions take place, even independently one from the other:
 a) the tray is of polymeric material;   b) the electrostatic field with vertical orientation is generated between two flat and parallel metal foils connected to a high voltage generator;   c) said electrostatic field has an intensity comprised between 150 and 200 kv/m, or more preferably between 160 and 180 kv/m;   d) the time of interposition of the tray in the electrostatic field is at least 5′;   e) the partial exsiccation of the collagen gel takes place at a constant temperature comprised between 10 and 40° C., preferably between 20 and 30° C., even more preferably between 22-28° C., or even more preferably between 24-26° C., in an aerated incubator without turbulence, up to a residual water content comprised between 70% and 30%.   
   
   
       17 . The process according to  claim 10  wherein the step of exiccation of the last collagen gel layer is carried out at constant temperature and in absence of ventilation. 
   
   
       18 . The process according to  claim 10  wherein the exsiccation takes place up to a reduction of the collagen gel thickness comprised between 30-70% compared to the thickness of the volume that was initially poured, preferably 40-60%, even more preferably about 50%. 
   
   
       19 . Process according to  claim 10 , wherein said tray is of polymeric material: polystyrene, polyethylene terephthalate (PET), polytetrafluoroethylene (teflon), polyvinyl chloride (pvc) etc. 
   
   
       20 . Process according to  claim 19  wherein the tray is made of polystyrene. 
   
   
       21 . Process according to  claim 19  wherein the thickness of the poured collagen gel layer is from 1.5 to 20 mm. 
   
   
       22 . Process according to  claim 10  wherein the collagen gel consists of type I, type II and/or type III collagen, or is a mixture of one or more types of collagen. 
   
   
       23 . Process according to  claim 22  wherein said mixture of one or more types of collagen is a mixture of type I collagen and type II collagen. 
   
   
       24 . Process according to  claim 23  wherein said mixture comprises type II collagen in a proportion comprised from 0.2 to 0.6% (w/v). 
   
   
       25 . Process according to  claim 24  wherein type II collagen is in a proportion comprised from 0.3 to 0.5% (w/v). 
   
   
       26 . Process according to  claim 10  wherein the collagen layer that is poured consists of a collagen gel mixture comprising type II collagen gels. 
   
   
       27 . Process according to  claim 26  wherein the type II collagen gel is obtained by treating a tissue, source of type II collagen, with NaOH according to a weight ratio between grams of starting tissue and grams of NaOH not higher than 3.5, preferably from 1.8 to 2.8. 
   
   
       28 . Process according to  claim 10  wherein the poured collagen gel comprises type I collagen gel. 
   
   
       29 . Process according to  claim 28  wherein the type I collagen gel is obtained by treating a tissue, source of type I collagen, with NaOH according to a weight ratio between grams of starting tissue and grams of NaOH from 0.05 to 0.5, preferably from 0.1 to 0.3, even more preferably from 0.12 to 0.18, or is 0.15. 
   
   
       30 . A multimicrolamellar membrane obtainable by the process according to  claim 10 . 
   
   
       31 . Membrane according to  claim 1  having a collagen content from 6 to 60 mg/cm 2 , preferably from 9 to 30 mg/cm 2  and preferably from 15 to 25 mg/cm 2 . 
   
   
       32 . Membrane according to  claim 1  having a yielding point value from 0.005 to 50 Mpa. 
   
   
       33 . Membrane according to  claim 32  wherein the yielding point value is comprised from 0.01 to 0.1 or from 0.5 to 50, more preferably from 2.5 to 15. 
   
   
       34 . Membrane according to  claim 1  having a Young modulus comprised from 0.001 to 100 Mpa. 
   
   
       35 . Membrane according to  claim 34  wherein the Young modulus is comprised from 0.01 to 0.1 Mpa or alternately from 0.5 to 60, more preferably comprised from 5 to 40. 
   
   
       36 . A method of tissue reconstruction, wherein said tissue is selected from the group consisting of: osteo-cartilaginous tissue, a tendon a bone or a joint, wherein the membrane according to  claim 1  is used. 
   
   
       37 . (canceled) 
   
   
       38 . A method according to  claim 36  comprising a step of growing and/or differentiating non-embryonic stem cells. 
   
   
       39 . (canceled) 
   
   
       40 . A method according to  claim 37  wherein said non-embryonic stem cells are suitable to differentiate into cells of the osteochondral lineage. 
   
   
       41 . A method according to  claim 37  wherein said non-embryonic stem cells are grown or differentiated in vitro.